Proliferation is the culmination of a cell's progression through the cell cycle resulting in the division of one cell into two cells. The five major phases of the cell cycle are G0, G1, S, G2, and M. During the G0, phase, cells are quiescent. Most cells in the body, at one time, are in this stage. During the G1 phase, cells, responding to signals to divide, produce the RNA and the proteins necessary for DNA synthesis. During the S-phase (SE, early S-phase; SM, middle S-phase; and SL, late S-phase) the cells replicate their DNA. During the G2 phase, proteins are elaborated in preparation for cell division. During the mitotic (M) phase, the cell divides into two daughter cells. Alterations in cell cycle progression occur in all cancers and may result from over-expression of genes, mutation of regulatory genes, or abrogation of DNA damage checkpoints (Hochhauser D., Anti-Cancer Chemotherapeutic Agents, 8:903, 1997).
Apoptosis or programmed cell death is the physiological process for the killing and removal of unwanted cells, and a mechanism whereby chemotherapeutic agents kill cancer cells. Apoptosis is characterized by distinctive morphological changes within cells that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al., Int. Rev. Cytol., 68: 251, 1980). The translocation of phosphatidylserine from the inner face of the plasma membrane to the outer face coincides with chromatin condensation and is regarded as a cellular hallmark of apoptosis (Koopman, G. et al., Blood, 84:1415, 1994). The mechanism of apoptosis is known to be mediated by the activation of a family of cysteine proteases, known as caspases.
Caspases recognize three major peptide sequences as substrates (Thornberry et al., J. Biol. Chem. 272:17907, 1997): (i) Tyr-Val-Ala-Asp (YVAD, caspase-1, -4), (ii) Asp-Glu-Val-Asp (DEVD, caspase-2, -3 and -7), and, (iii) Ile-(Leu)-Glu-X-Asp (I(L)EXD; caspase-8 and -10). Sequence recognition in a protein target results in a limited and specific proteolysis of the target, such as activation of caspase-7 by caspase-3, degradation of structural protein targets including, but not limited to, lamins, or activation of enzymes including, but not limited to, poly(ADP-ribose) polymerase. Caspase-3 was reported to be cleaved into its catalytically active subunits (17 and 13 kDa) following pro-apoptotic signals, leading to apoptosis (Susin et al., J. Exp. Med. 186:25, 1997).
During apoptosis, the activation of caspases results in proteolytic cleavage of numerous substrates. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis (O'Brien et al., Biotechniques 30:886, 2001).
The extracellular matrix (ECM) impacts behavior of normal and tumor cells (Radisky et al., Seminars Cancer Biol., 11:87, 2001). Therefore, the interaction between tumor cells and the ECM components, when tumor cells are plated on the ECM, will activate signal transduction events mimicking several biopathological characteristics of tumors in vivo, such as modulation of cell-cell contacts (Weaver et al., J. Cell Biol., 137:231, 1997).
Synthetic oligonucleotides are polyanionic sequences that are internalized in cells (Vlassov et al., Biochim. Biophys. Acta, 11197:95, 1994). Synthetic oligonucleotides were reported that bind selectively to nucleic acids (Wagner, R., Nature, 372:333, 1994), to specific cellular proteins (Bates et al., J. Biol. Chem., 274:26369, 1999) and to specific nuclear proteins (Scaggiante et al., Eur. J. Biochem, 252:207, 1998) in order to inhibit proliferation of cancer cells.
Synthesis and physical properties of oligonucleotides with a cholesteryl moiety have been described. The attachment of a cholesteryl moiety to the 3′-end of antisense oligonucleotides enhances their activities (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553, 1989; Boutorin et al., FEBS Letter, 254:129, 1989; Corrias et al., J. Neurooncol. 31:171, 1997; U.S. Pat. No. 4,958,013; WO Patent No. 9714440). The attachment of a cholesteryl moiety to the 3′-end enhances the uptake of antisense molecules by cells (Corrias et al., J. Neurooncol. 31:171, 1997), and increases antisense vascular retention in vivo (Fleser et al., Circulation 92:1296, 1995). Internucleoside cholesteryl side chains linked to phosphorous via phosphoramidate bonds have been described as a modification to increase the activity of antisense molecules (U.S. Pat. No. 4,958,013). Homopolymers of 15 cytidine or thymidine residues with a cholesteryl moiety at the 5′-end were found to modulate cytosolic Ca2+ levels in pro-myelocytic leukemia cells, while heteropolymeric sequences with a cholesteryl moiety at the 5′-end or cholesteryl-modified phosphorothioate sequences were inactive (Saxon et al., Antisense Res. Dev. 2:243, 1992). Heteropolymers consisting of 15 phosphorothioate deoxynucleotides with alternating cytosine and adenosine residues, or homopolymers with 15 cytosine or thymidine residues, were shown to be potent inhibitors of methotrexate transport when a cholesteryl group was linked to the 5′-end (Henderon et al., Nucl. Acids Res. 25:3726, 1995). The covalent modification of a 10 base homocytidine phosphorothioate oligonucleotide with a cholesteryl moiety at the 5′-end blocked the formation of syncitia in T lymphocytes infected with HIV-1 or HIV-2 through inhibition of HIV reverse transcriptase (Stein et al., Biochemistry 5:2439, 1991).
The attachment of a cholesteryl moiety to oligonucleotides has minimal effects on the growth of cancer cells (Henderon et al., Nucl. Acids Res. 25:3726, 1995). Typical features of apoptotic cell death were not observed in cancer cell lines treated with 3′-end cholesteryl oligonucleotides (Corrias et al., J. Neurooncol. 31:171, 1997).
Most anti-cancer therapies, whether directed to inhibition of proliferation, induction of cell cycle arrest, induction of apoptosis, stimulation of the immune system or modulation of extracellular matrix-cell interaction have proven to be less than adequate for clinical applications. Many of these therapies are inefficient or toxic, have significant adverse effects, result in development of drug resistance or immunosensitization, and are debilitating for the recipient
Therefore, there is a continuing need for novel compositions and methods that induce cell cycle arrest in cancer cells, that induce apoptosis in cancer cells, and that modulate extracellular matrix-cell interactions.